Previous studies of the dynamics and functional relationships of genes and the networks they comprise followed gene expression after a well-defined perturbation to a culture growing in steady state. Traditionally, such perturbations involved a pulse of preferential carbon source, heat or nutrients (RONEN and BOTSTEIN 2006). However, when one wants to perturb only a single element of the system, such as a transcription factor, use of the traditional perturbation methods result in side effects on physiology and gene expression that can be deleterious to the experiment. What is needed to resolve this problem is finding specific ways of turning on (or off) only a single transcription factor.
Significant effort in the prior art had been placed on achieving tightly controlled gene expression systems across a spectrum of organisms, guided by the transformative discoveries of gratuitous inducers of expression of the lactose operon genes in E. coli. The significant desirable features of a useful induction system include (1) a complete lack of target gene activation in the absence of inducer, (2) rapid induction of a target gene in the presence of inducer, and (3) the availability of a truly gratuitous inducer. A gratuitous inducer should interact with no other regulatory system and should be essentially inert with respect to physiology.
A common approach in mammalian cells is a direct adaptation of bacterial systems: tetracycline-inducible tetR-based systems (LABOW et al. 1990). By fusing activating domains (e.g., VP16) to tetR and expressing this chimeric transcription factor, transcription of a target gene downstream of a minimally active promoter with the appropriate operator sites can be maintained at high levels in the absence of inducer. TetR-VP16, also called tTA (tetracycline transactivator), unbinds DNA in the presence of tetracycline, thereby repressing transcription. This configuration is referred to as tet-off. Tet-off has been used for studying the effects of depletion of a target gene, but the kinetics of transcriptional shut off do not result in rapid elimination of the protein products of the regulated genes, as many proteins have long half-lives. Therefore, a better method for rapid depletion of a target gene is needed.
The most widely used overexpression system in S. cerevisiae has utilized GAL4-mediated induction of targets, which are placed downstream of promoters containing 17-mer UASgal sites (such as PGAL1, PGAL7, PGAL10), CGG-N11-CCG (SEQ ID NO: 1) (GINIGER et al. 1985). The UASgal motif is recognized by dimerized GAL4 proteins (HONG et al. 2008; MARMORSTEIN et al. 1992). Deletion of GAL1 has made galactose overexpression of GAL-driven targets nearly gratuitous (HOVLAND et al. 1989). Since GAL genes are catabolite repressed, activation required that cells be grown in relatively poor, non-glucose carbon sources prior to the galactose pulse.
A more convenient system was developed that avoids the necessity of switching the carbon source. A chimeric transcriptional activator called GEV (LOUVION et al. 1993) contains the DNA binding domain of Gal4p, the hormone binding domain (HBD) of the human estrogen receptor (hER), and the highly electronegative portion of herpes simplex virus protein VP16, which confers strong transcriptional activity (SADOWSKI et al. 1988). Though GEV binds DNA via a GAL4 DNA binding domain, it is not subject to inhibition or repression by glucose, making it feasible to conduct induction and overexpression experiments in standard glucose-containing media simply by the addition of the inducer β-estradiol. The presence of the HBD provides a simple on/off switch for GEV activity; steroid receptor binding of ligand results in a conformational change of the receptor and its subsequent disassociation from the Hsp90 chaperone complex (PRATT and TOFT 1997).
Previously, improvements over the original GEV system were made by placing GEV expression under the control of low-strength constitutive promoters to reduce errant expression of target genes in the absence of β-estradiol (GAO and PINKHAM 2000; VEATCH et al. 2009). Another GEV-based induction system implemented an autocatalytic approach to rapidly increase GEV production in the presence of β-estradiol. In this case, GEV was placed under the control of the GAL1 promoter, which contains 4 UASgal sites (QUINTERO et al. 2007). However, the prior art only envisioned GEV for induction of gene expression.